Cloning,Expression Of J3 Gene And Its Mechanism Regulation In Flowering Time Control Of Brassica Juncea

Brassica juncea is an important vegetable crop widely planted in southwest China.And the yield and quality of their products are seriously affected by flowering time.The floral transition is a significant developmental stage from vegetative to reproductive in flowering plants.The main factors influencing the flowering time of B.juncea include the external environmental cues such as photoperiod and temperature,as well as the internal conditions such as plant growth state,developmental stage and genetic factors.There are four complex genetic pathways to regulate flowering including long-day photoperiod,low-temperaturevernalization,autonomousandgibberellin（GA）-dependent pathways.Although each of these four pathways has a different genetic network regulation,it ultimately focuses on the same flowering signal integrator.At present,many regulators involved in flowering control have been identified through molecular genetics studies in Arabidopsis.Among them,MADS-box transcription factors play crucial roles in promoting or inhibiting flowering,such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1（SOC1）、AGAMOUS-LIKE24（AGL24）.DNAJ HOMOLOG3（J3）is a new flowering regulatory factor,which regulates the expression of FT and SOC1 by interacting with SVP in Arabidopsis.However,it is not clear whether J3 directly regulates the flowering time of SOC1 and AGL24 in B.juncea.In this study,B.juncea J3（BjuJ3）gene was cloned and its expression pattern was investigated.Also,we examined protein-protein interactions of BjuJ3 with BjuSOC1and BjuAGL24 via yeast two-hybrid and pull-down,as well as protein-DNA interactions of BjuJ3 with promoters of BjuSOC1 and BjuAGL24 via yeast one-hybrid and Dual-Glo?Luciferase.The results are as follows:1.Cloning and sequence analysis of BjuJ3 geneThe total RNA was extracted from the shoot of B.juncea seedlings.The cDNA was obtained by reverse transcription and used as a template for amplifying BjuJ3 gene via PCR.Then we got BjuJ3 gene of 1275 bp which encoded 425 amino acids.Phylogenetic tree analysis showed that BjuJ3 shared high similarity in amino acid sequence with other Brassica species in the same branch.2.BjuJ3 promotes flowering in tobaccoTo elucidate the function of BjuJ3 in the regulation of flowering time,we created BjuJ3 transgenic tobacco lines by agrobacterium-mediated method.The results suggested that BjuJ3 could promote flowering and was responsible for the early-flowering phenotype.3.BjuJ3 expression is regulated by photoperiod and vernalizationThe qRT-PCR assays showed that BjuJ3 was ubiquitously expressed in roots,stems,leaves,flower buds and open flower organs,with the lowest expression in flower buds.The BjuJ3 expression was consistently higher in the LDs and 4℃than that of the normal conditions.These results supported that BjuJ3 expression was induced to promote flowering upon LDs and vernalization.4.Protein-protein interactions of BjuJ3 with BjuSVP,BjuSOC1 and BjuAGL24The yeast recombinants vectors pGADT7-J3 and pGBKT7-J3 were used as yeast bait proteins to perform yeast two-hybrid assay,respectively.Then the yeast zygotes were plated on SD∕-Ade∕-His∕-Leu∕-Trp∕Xa-Gal medium and found that BjuJ3∕BjuSVP,BjuJ3∕BjuSOC1 and BjuJ3∕BjuAGL24 failed to grow on the media,indicating that BjuJ3 could not interact with BjuSVP,BjuSOC1 and BjuAGL24.Furthermore,pET32a（+）-J3 was used as the bait protein in the GST pull–down assay and found that the magnetic beads did not adsorb pGEX-BjuSVP,pGEX-BjuSOC1 and pGEX-BjuAGL24.It suggested that BjuJ3 could not bind to BjuSVP,BjuSOC1 and BjuAGL24.5.Protein-DNA interactions of BjuJ3 wtih promoters of BjuSOC1 and BjuAGL24In yeast one-hybrid assays,the Y1HGold[（pAbAi-SOC1）×（pGADT7-J3）]could grow on SD∕-Leu∕AbA¹⁰⁰ medium,whereas the Y1HGold[（pAbAi-AGL24）×（pGADT7-J3）could not grow on SD∕-Leu∕AbA³⁵⁰ medium,indicating that BjuJ3 protein only interacted with BjuSOC1 promoter.Additionally,the pGreen II 62SK-BjuJ3 was respectively fused with pGreen II 08 00LUC-SOC1 and pGreen II 0800LUC-SOC1-1 in Dual-Glo?Luciferase assay,The results showed that the ratio of renilla fluorescence to firefly fluorescence was significantly higher than that of the negative control and other treatments,indicating that BjuJ3 indeed interacted with the promoter of BjuSOC1.